The red blood cell count, white blood cell count and the determination of blood cell indices are the most frequent of all clinical laboratory tests performed on patients in doctors' offices, clinics and hospitals in the United States and throughout the world. These tests provide essential information as to the health characteristics of individuals of all age groups.
Processes for evaluating and testing blood which are in widespread use in the art today uniformly require a sample of blood to be removed from the body for testing and analysis. The procedure for taking blood, known as phlebotomy, ordinarily involves the removal of blood from capillary or peripherial blood (usually in infants) and venous blood. Only certain licensed persons are permitted by law to carry out this procedure. These include nurses, laboratory technicians, physicians, and other specially trained persons.
In infant phlebotomies, the sample is ordinarily taken from the palmar surfaces of the tip of a finger or the plantar surfaces of the great toe or heel. The site is first rubbed vigorously with a gauze pad moistened with seventy-percent alcohol to remove dirt and debris and to increase blood circulation. Once the skin has dried, a puncture 2-3 mm deep is made with a blade or lancet. After the sample has been collected, slight pressure is applied to the area of the puncture with a gauze pad. This procedure is usually painful and frightening to the infant which results in further difficulties in effecting the phlebotomy.
The most common form of adult patient phlebotomy involves the removal of venous blood through puncture of a vein in the patient's arm. This procedure is also somewhat painful and is viewed with apprehension by most patients. Veins must be carefully inspected, particularly with those patients who have already had numerous punctures. The patient's life may depend on vein patency, and care must be taken to preserve these vessels. Hematomas or ecchymoses are usually evidence of the operator's poor technique or judgment.
The equipment necessary for this procedure ordinarily includes a syringe and a needle of the appropriate diameter. This needle must be carefully inspected as a blunt or bent tip will damage the patient's vein and often lead to failure to collect blood from the vein. A tourniquet is also used in the procedure in order to make the veins more prominent and help to eliminate blind probing.
The procedure involves making the patient comfortable, preferably having them sit in a chair with the patient's arm accessible to the operator. The tourniquet is then applied and the skin surrounding the target site is cleaned with a seventy-percent alcohol solution and permitted to dry. Once the appropriate vein has been located, the needle is pushed into the vein with a single direct puncture of both the skin and the vein. The tourniquet is then loosened and the desired amount of blood is obtained. The operator must be aware of the patient at all times as the trauma of venipuncture can cause the patient to faint. After the procedure is completed the operator must also insure that the patient's condition is satisfactory before he is dismissed.
Complications of venipunctures do occur and include a measurable increase in the concentration of blood cells when the tourniquet is applied for periods greater than sixty seconds. Failure of the blood to enter the syringe may also occur. This may result from excessive pull on the plunger of the syringe which can cause the vein to collapse. The piercing of the outer coat of the vein without entering the lumen can also account for failure. These complications are occasionally followed by hematoma formation. When this occurs, the needle must be withdrawn and the procedure must be carried out on the other arm. Late complications include possible thrombosis of the vein due to trauma, especially following many venipunctures at the same site. Finally, where non-disposable or contaminated needles are used, the transmission of contagious diseases such as serum hepatitis, etc. may be effected.
Blood removed from the patient's body is usually transported to a clinical laboratory where it is tested and the results are sent back to the requesting physician. In most doctors' offices in the Untied States today, such tests are performed at central laboratories and the results are usually available the following day. In addition to the delay, there is also a substantial cost for such testing because of the logistics involved in specimen collection, transport, laboratory accessioning, skilled and licensed personnel required for the removal procedure and testing of the specimens, reagents utilized and expensive instrumentation employed. Further disadvantages of this in vitro method include the numerous sources of error introduced into the analysis of the specimen as a result of in vitro changes in the blood sample. For example, in blood kept at room temperature, swelling of the red blood cells between six and twenty-four hours raises the hematocrit and mean corpuscular volume (MCV) and lowers the mean corpuscular hemoglobin concentration and the red blood cell sedimentation rate.
Current in vitro testing requires strict adherence to procedure. Before taking a sample from a tube of venous blood for hematologic determination, it is important that the blood be mixed thoroughly. If the tube has been standing, this requires at least sixty inversions of the tube or two minutes on a mechanical rotator; less than this leads to unacceptable deterioration in precision.
The most common tests performed on blood samples taken from patients are the hematocrit (Hct), or the hemoglobin (Hb), which are often used interchangably, depending upon the individual preference of the treating physician. They are used to determine anemia, to monitor conditions in which the blood loss occurs, chronic diseases, drug reactions, allergies, and the course of therapy.
The Hct of a sample of blood is defined as the ratio of the volume of erythrocytes (red blood cells) to that of the whole blood. It is expressed as a percentage or, preferably, as a decimal fraction. The units (L/L) are implied. The venous hematocrit agrees closely with the hematocrit obtained from a skin puncture; both are greater than the total body hematocrit.
Hemoglobin, the main component of the red blood cell, is a conjugated protein that serves as a vehicle for the transportation of oxygen and CO.sub.2, throughout the body. When fully saturated, each gram of hemoglobin holds 1.34 ml of oxygen. The red cell mass of the adult contains approximately 600 g of hemoglobin, capable of carrying 800 ml of oxygen. The main function of hemoglobin is to transport oxygen from the lungs, where oxygen tension is high, to the tissues, where it is low. As used in this application the term hemoglobin (Hb) refers to the concentration of the iron-containing protein pigment found in red blood cells.
The Hct and Hb are often provided along with the total red blood cell count (RBC) which is usually expressed in the form of a concentration--cells per unit volume of blood. Once these three values are known (Hct, Hb and RBC), three red blood cell indices are calculated. These indices are particularly useful in the morphologic characterization of anemias. These values include the mean cell volume (MCV) which is the average volume of red blood cells and is calculated from the Hct and the RBC. Utilizing the formula: EQU MCV=Hct.times.1,000/RBC (in millions per u)
The mean cell hemoglobin (MCH) may also be calculated and is the content of Hb in the average red blood cell; it is calculated from the Hb concentration and the RBC utilizing the following formula: ##EQU1##
Another index calculable from the Hb and Hct is the mean cell hemoglobin concentration (MCHC). This index is the average concentration of Hb in a given volume of packed red blood cells. It is calculated using the following formula: ##EQU2##
Other characteristics of red blood cells which are available utilizing today's testing methods include values for the variability of the MCV about a mean value and estimates of abnormality in red blood cell morphology.
The above described indices are discussed in much greater detail in John Bernard Henry, M.D., Clinical Diagnosis And Management By Laboratory Methods, Part IV (17th edition 1984).
Modern clinical laboratory instrumentation has been built to make these primary analyses simultaneously in vitro on blood samples removed from the patient and the calculated indices are readily produced by these instruments. The calculated indices are often the preferred data on which physicians base their conclusions about a patient's condition.
A large number of testing methods, instrumentation, and techniques have been used in measuring and approximating values for Hct, Hb and RBC. The most common method used to determine the Hct (the ratio of packed red blood cells to volume of whole blood) involves centrifugation wherein a given blood sample is placed into a centrifuge for five minutes at approximately 10,000 to 12,000 g. The volume is then calculated by measuring the level of the red blood cells as a ratio of the total volume.
Sources of error in this method include insuring that the sample is subject to adequate centrifugal force for a sufficient duration so that the red cells may be packed and give an accurate reading. In addition, the final value must be corrected for trapped plasma present within the packed red blood cells. Technical errors in this method include failing to mix the blood adequately before sampling, improper reading of the level of cells to plasma, and irregularity of the inside diameter of the specimen tubes.
Methods used in the art to determine the Hb in a sample of blood include the cyanmethemoglobin method, the oxyhemoglobin method and the method of measuring iron content of the sample. Of the above three methods, the first (the cyanmethoglobin method) is recommended by the International Committee for Standardization in Hematology. That method involves diluting a sample of blood in a solution of potassium ferricyanide and potassium cyanide. The potassium ferricyanide oxidizes hemoglobins and potassium cyanide provides cyanide ions to form hemiglobincyanide which has a broad absorption maximum at a wavelength of approximately 540 nm. The absorbence of the overall solution can then be measured in a photometer or spectrophotometer at 540 nm and compared with that of a standard hemiglobincyanide solution.
The oxyhemoglobin method is not widely used, however it does yield reproducible results. The main disadvantage however is the lack of a stable standard with which to compare the results. This method involves the creation of a 1:251 dilution of blood in 0.007 N NH.sub.4 OH utilizing distilled water. This solution is then shaken to insure proper mixing and oxygenation of the hemoglobin. The solution is read in a photometer with a green filter with a 0.007 N ammonium hydroxide solution used as a standard.
The last method listed above involves a procedure whereby Hb may be measured by determining the iron content of the whole blood. The non-hemoglobin iron in blood is negligible compared to hemoglobin iron, however, the iron must first be separated from the hemoglobin, usually by acid or by ashing. It is then either titrated with TiCl.sub.3 or complexed with a reagent to develop color that can be measured photometrically since the iron content of hemoglobin is given as 0.347 percent, the concentration of hemoglobin in blood is calculated by dividing the iron concentration by 3.47.
Numerous sources of error are present in the above methods including those of the sample, the method, the equipment, and/or the operator. Errors inherent in the sample include improper venipuncture technique which may introduce hemo concentration, which will make hemoglobin concentration and cell counts too high. The photometer used for determining Hb must be calibrated in the laboratory before its initial use and must be re-checked frequently. The wavelength settings, filters and meter readings require constant monitoring.
The RBC, or red blood cell count may be determined by a number of different cell counting procedures. Any of these procedures includes three steps: dilution of the blood, sampling the diluted suspension into a measured volume, and counting the cells in that volume. Optical and electronic equipment have been developed to perform red blood cell counting, inter alia, in flowing systems in vitro. This equipment can also separately measure Hb by a chemical method in a separate analytic channel. The most widely used of such instruments is manufactured by Coulter Diagnostics of Hialeah, Florida, wherein red blood cells are passed through a glass tube with an aperture such that single cells can be detected based on a decrease in the voltage between electrodes positioned in a constricted portion of the tube. This measurement of voltage decrease yields an indirect value for RBC. The MCH is then computed utilizing the relationship: EQU MCH=Hb/RBC
then the Hct is calculated from the relationship: EQU Hct=MCV.times.RBC
after MCV is indirectly derived from the mean height of the voltage pulses formed during the red blood cell count.
Another instrument, made by Technicon Instrument Co., of Tarrytown, N.Y., and Fisher Scientific Co., of Pittsburgh, Pa., generates a value for RBC by using a system wherein red blood cells flowing in glass tubes are counted by deflection (scattering) of a beam of light that is directed to an opposing photo multiplier. Hb is measured by a chemical method in a separate channel of the instrument. The above-described instruments and methods are all characterized by their in-vitro analysis of specimen of blood withdrawn from the patient's body without the use of image analysis and yielding indirect (calculated or estimated) values for red blood cell indices.
Image analysis has been utilized to examine stained smears of blood fixed to glass slides with computer-controlled microscopes. Examples of such instruments include the "Hematrak" instruments manufactured by the Geometric Data Corp., Wayne, Pa. These instruments utilize pattern recognition software which identifies each of the major classes of white blood cells according to size, shape, staining and other morphologic characteristics. They also identify red blood cells for their morphology.
The imaging system includes a microscope with an automated scanning stage and an automatic focusing objective. Filters split the light transmitted through the stained cell into red, blue, and green portions of the spectrum. These allow the staining properties of the portions of the cells to be characterized. These instruments, however, do not produce any quantitative measures of red blood cells. These, and similar instruments, are also characterized by their in-vitro analysis of stained blood, fixed to slides for analysis. None perform quantitative analysis of red blood cell parameters.
Instruments which utilize flowing systems in conjunction with image analysis are exemplified by the International Remote Imaging Systems (IRIS) instruments which are designed to analyze in-vitro stream of the specimen flowing through glass tubing. This system utilizes a flow analyzer and system described in U.S. Pat. No. 4,338,024, issued to Bolz, et al., and assigned to IRIS. In that system, urine specimens are aspirated through a flow cell which is designed to orient cells and particles in one plane. Moving particles and cells are then photographed by a high-speed camera and counted by a microprocessor programmed to identify and classify the different particles and cells, including red blood cells. No quantitative analysis of red blood cell parameters is performed and the spectral analysis utilized is by transmission, rather than reflectance spectrophotometry.
A video computer system for measuring the lineal density of red blood cells and capillaries in vivo is described in an article by C. G. Ellis, et al. in Microvascular Research 27, Pages 1-13 (1984). This system utilizes a computerized frame-by-frame analysis of video images in order to perform continuous measurements of lineal density based on the spatial-average of blood opacity over a selected length of capillary. This method does not attempt to measure or even detect individual RBCs, but rather is directed to the measurement of light intensities along the centerline of the image of a capillary such that the number of RBCs in a given length of capillary is inversely related to he average light intensity over that length. A light intensity profile is determined utilizing a video analyzer which, for each frame, measured the light intensity values along a given length of capillary, first in the absence of RBCs to determine the "background light intensities" and then as the flow of RBCs is reestablished. A plot of mean opacity versus RBC for a particular capillary segment is then plotted and thus a value for RBC is estimated given a particular measured mean opacity. This system does not measure Hb, Hct or any of the other indices directly.